人椎间盘髓核来源的诱导多能干细胞重编程及功能的研究  

Generation and Functional Characterization of Induced Pluripotent Stem Cells From Human Intervertebral Disc Nucleus Pulposus Cells

作　　者：吴张松 罗志强[1] 张明裕 廉翠红[2] 林欣燕 梁雨虹 顾洪生[2] 朱艳霞[1] 

机构地区：[1]深圳大学医学部,深圳518060 [2]深圳大学附属第一医院脊柱外科,深圳518060

出　　处：《生物化学与生物物理进展》2017年第7期591-600,共10页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(81301597);深圳新兴战略产业发展专项资金(JCYJ20150525092940984/JCYJ20160422090807181)资助项目~~

摘　　要：椎间盘退变始发于髓核组织,获得足够有功能的髓核细胞是研究及治疗椎间盘退变的关键.而人诱导多能干细胞(induced pluripotent stem cell,iPSC)不仅为建立疾病模型以研究疾病发生发展机制开辟了道路,还在再生医学领域展现出了广阔的应用前景.我们首先从椎间盘退变患者微创手术获得的髓核组织内分离髓核细胞,将携带OCT3/4、SOX2、KLF4和c-MYC的仙台病毒(Sendai virus,Se V)转染髓核细胞,重编程获得iPSC.通过检测多能细胞特异性标志、体内成瘤实验、甲基化及核型分析对所获得的iPSC进行鉴定.并以皮肤成纤维细胞来源iPSC作为对照,在二维和三维水凝胶中对iPSC进行定向分化,检测髓核细胞相关蛋白和基因的表达,比较分析2种iPSC向髓核细胞的分化效率.结果显示,iPSC能表达多能细胞特异性标志,具有正常的二倍体核型,畸胎瘤实验显示三个胚层的出现.诱导分化后的iPSC表达髓核相关基因和蛋白,在水凝胶中诱导培养后,iPSC表达更多的髓核相关基因和蛋白.髓核来源的iPSC与成纤维细胞来源的iPSC相比,可表达更多的髓核相关基因和蛋白.本研究首次将患者退变髓核细胞重编程成iPSC,并在水凝胶内将其诱导分化为髓核样细胞,为椎间盘退变个体化细胞治疗奠定基础.Intervertebral disc degeneration is believed to originate in the nucleus pulposus（NP） region, it is important to obtain greater numbers of active NP cells for the study and therapy of disc degenerative disease（DDD）. Human induced pluripotent stem cells（iPSCs） are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. We isolated NP cells from DDD patients with our improved method, and reprogramed primary NP cells into iPSCs with Sendai virus vectors encoding 4 factors. Successful reprogramming of iPSCs was verified by specific surface markers and teratoma, and differentiation of iPSCs into NP-like cells was performed in culture plate and hydrogel, with skin fibroblast derived-iPSC used as control. It was demonstrated that iPSCs derived from NP cells were featured with normal karyotype, and showed expression of pluripotency markers and were able to form teratoma in nude mice. NP induction of iPSCs resulted in their expression of NP cell specific matrix proteins and related genes. NP derived-iPSCs without induction also showed a basal level of expression of some NP-like phenotype characteristics. What＇s more, NP derived-iPSCs prefer to differentiate into NP-like cells in hydrogel rather than in culture plate.This is the first protocol for generation of iPSCs from NP cells of DDD patients, and we successfully differentiated this iPSCs into NP-like cells in hydrogel. This method opens a window in the treatment of DDD by using patient-specific NP cells in a relatively simple and straight forward manner.

关 键 词：椎间盘退行性疾病 髓核 诱导多能干细胞 重编程 分化 

分 类 号：R3[医药卫生—基础医学] Q2[生物学—细胞生物学]